Molecular Processes-gene Regulation

8/6/2019 Molecular Processes-gene Regulation

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We must not hide from ourselves

the fact that the causal investigationof organisms is one of the most

difficult, if not the most difficult

problem which the human intellecthas attempted to solve, since every

new cause ascertained only gives

rise to fresh questions regarding thecause of this cause. Wilhelm Roux


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Most genes code for protein products; a few

are RNA transcripts.

Names of genes are written italicized inlower case, while the protein product in

Roman type (e.g. thewingless gene codes

for the Wingless protein).

The genome of a cell contains in its DNAsequence the information to make many

thousands of different protein and RNA

molecules.

Both embryonic and differentiated cells

contain all the genetic instructions

necessary to direct the formation of a

complete organism.


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How does a fertilized egg cellknow what kind of

organ/organism to become?How does a cell determine

which gene product tosynthesize at any given time?


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1.ENHANCERS ARE MODULAR. There arevarious DNA elements that regulate temporal

and spatial gene expression, and these can bemixed and matched. Whether the gene is

transcribed or not will depend on the

combination of transcription factors present.


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Does thisalso mean

that

enhancers

can direct

expression

ofany

gene

sequence,

orwhenplaced in

new

locations?


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Activation of regulatory genes such as Pax6 in new places

Using Drosophila embryos, GAL4 transcription factor was placed

next to an enhancer for genes normally expressed in the developing

antennae. Therefore, GAL4 should also be expressed in antennal

tissue. A second transgenic fly, placed the cDNA for the Drosophila

Pax6 gene downstream from a sequence composed of five GAL4-binding sites. In flies in which the Pax6 gene was expressed in the

incipient antennal cells, part of the antennae gave rise to eyes. It

appears that in Drosophila, Pax6 not only activates those genes that

are necessary for the construction of eyes, but also represses those

genes that are used to construct other organs.


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The enhancer trap technique. (A) A reporter gene is fused to a weak promoter that

cannot direct transcription on its own. This recombinant gene is injected into thenucleus of an egg and integrates randomly into the genome. If it integrates near an

enhancer, the reporter gene will be expressed when that enhancer is activated,

showing the normal expression pattern of a gene normally associated with that

enhancer. (B) Reporter gene expression (dark region) in a Drosophila embryo

injected with an enhancer trap. This expression pattern demonstrated the presence

of an enhancer that is active in the development of the insect nervous system and

which was unrecognized before the procedure.

How are

enhancers

detected?


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The c-myc gene synthesizes veryshort-lived mRNA and

protein products when stimulated bya varietyof growthfactors, appearing suddenlyas cells are induced from

the G0 state into G1, and are degraded shortlythereafter.

The c-myc proteins

signal cell division,

and if theyare notdegraded rapidly, the

cell will continue

proliferating, thereby

increasing the risk of

tumor formation. The translocation of

the c-myc gene to an

IgG enhancer in a

single cell can cause

a form of leukemia called Burkittlymphoma.


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First discovered in

Drosophila; mice and

humans contain at least

38 Hox genes arranged

in 13 paralogous groups

in 4 different

chromosomes.

2. FAMILIESOF TRANSCRIPTION FACTORS

bind to the enhancer or promoter regions

for specific gene expression

a. Homeodomain factors- genes coding for

proteins with a highly conserved homeobox of

61 amino acids (183 nucleotides) implicated in

cranio-caudal segmentation of the body.


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Hox gene mutations in mice have been shown to

produce homeotic transformation of vertebral or

spinal segments.

HOX13 mutations in man

cause synpolydactyly

(interphalangeal

webbing and extra

digits in hands & feet). The fibroblast growth

factor (FGF) selectively

activates posterior

homeobox genes

Transforming growthfactor-F (TGF-F)

selectively activates anterior Hox genes

Retinoic acid cause morphological abnormalities by

posterization of Hox genes.


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b.LIM domain- cysteine-

rich zinc-binding

region responsible for

protein-protein

interactions.

Also shown to play

roles in cytoskeletal

organization, organdevelopment and

oncogenesis.

Absence of Lim

proteins results in

development ofheadless mammalian

embryos (Lim-1 in the

organizer and Islet-1

in motor neurons).


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c. Paired box- consist of 9 known

proteins (Pax1-9) consisting of

paired domain of128 aminoacids that bind to DNA, and

may also contain a

homeodomain (Pax6in the eye,

Pax3 in the developing somite).


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d. Zinc-finger

proteins- DNA-

binding region

contains

projections or

fingers with

Cys/His residues

folding around a

Zinc atom (WT-1

in the kidney,

Krox20 in the

rhombomeres of

the hindbrain).

The family

of zinc

finger

proteinsare named

after the

amino

acids that

grasp the

zinc.

Estrogen receptor


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The WT1 factor binds to the regulatory regions of

several genes that are active during kidney

development & inhibits the expression of certain

growth factors in the developing kidney

People with one mutant WT1 allele have urogenital

malformations and develop Wilms tumor of the kidney

Functional domains of zinc finger transcription

factors. Cysteine (C) and histidine (H) coodinate a zinc

atom, causing the looping out of the "zinc fingers."


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e. Nuclear receptor superfamily-O

n binding of the hormone (e.g.estrogen, progesterone, testosterone, cortisone, ecdysone,

nonsteroid lipids such as retinoic acid, thyroxine, and vitamin D),

the NR/hormone complex can form active dimers which can now

bind to target genes. The DNA sequences capable of binding

nuclear hormone receptors can be either be enhancers or

promoters.


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Active forms are

heterodimers containing abasic DNA-binding region

& a hydrophobic helix-

loop-helix region

responsible for proteindimerization (e.g., E12,

E47, myogenic factor

MyoD).

Those with HLH but not

the basic part forminactive dimers with other

bHLH proteins & inhibit

their activity (e.g., Id, a

myogenesis inhibitor).

f. Basic helix-loop-helix (bHLH) proteins


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j. POU- contains a homeobox, plus a region coding for

75 amino acids which bind to DNA through a helix-

loop-helix structure (Pit-1 activates the genes

encoding growth hormone, prolactin, & other

pituitary proteins; Oct1 & Oct2, activate IgG genes;

& UNC-involved in determining neuronal cell fates.


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k. Leucine-zipper- bind

DNA as dimers. A

leucine zipper is formedby two a helices, held

together by hydrophobic

interactions between

leucine residues, whichare located on one side

of each helix.

Examples include AP-1,

CREB, and Gcn4.The structure of the AP-1/DNA complex. AP-1 is a

dimer formed by Jun and its homologous protein Fos.

It contains a leucine zipper motif where two a helices

look like a zipper with leucine residues (green color)

lining on the inside of the zipper.


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3. SELECTIVE INHIBITIONOF MRNA

TRANSLATION

During the growth of the oocyte, mRNAs arestored in mRNP (message ribonucleoprotein)

complexes, localized within a specific region

of the cytoplasm, or homogeneously

dispersed within the cytoplasm of the entireoocyte. They are not translated until some

condition is satisfied.

Oocyte maturation inhibitor (OMI) prevents

translation of mRNAs unless cytoplasmalkalinization takes place after fertilization.

Similar mechanisms require specific signals

on the mRNA, typically located in the 3 UTR.


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4. CONTROL OF RNA EXPRESSION BY CYTOPLASMIC

LOCALIZATION

Selective localization of messages is

accomplished through their 3 UTRs. Certain mRNAs in Xenopus embryos

are selectively transported to the

vegetal pole of the frog oocyte. After

fertilization, these messages make

proteins that are found only in the vegetal blastomeres

In Drosophila, the bicoid & nanos messages are each

localized to different ends of the oocyte. This localization

allows the Bicoid protein to form a gradient wherein the

highest amount of it is at the anterior

pole, while the Nanos protein forms

a gradient with its peak at the posterior.

The ratio of these two proteins will

eventually determine the A-P axis of

the Drosophila embryo & adult.


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The bicoidandnanos mRNAs

in Drosophila egg are localized

near the anterior and

posterior poles, respectively.The caudal, hunchback, and

pumiliomRNAs are distributed

throughout the egg cytoplasm.

The gradients of Bicoid(Bcd)

andNanos proteins leadtoaccumulation of Hunchback

protein in the anterior and

caudal protein in the posterior

of the egg. Because Pumilio

protein requires Nanos proteinfor its activity as a

translational repressor of

hunchback, it functions only at

the posterior end.


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Molecular Processes-gene Regulation

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